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KMID : 0545120190290030373
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Journal of Microbiology and Biotechnology
2019 Volume.29 No. 3 p.373 ~ p.381
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Impact of Expanded Small Alkyl-Binding Pocket by Triple Point Mutations on Substrate Specificity of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase
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Dwamena Amos K.
Phillips Robert S.
Kim Chang-Sup
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Abstract
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Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency (k cat/K M) of the M151A/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3’-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using V115A/C295A/I86A mutant in asymmetric reduction. The A85G/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3’-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3’- methoxyacetophenone, with improved yield. In terms of thermal stability, the M151A/ C295A/I86A and V115A/C295A/I86A mutants significantly increased ΔT1/2 by +6.8ºC and +2.4ºC, respectively, with thermal deactivation constant (k d) close to the wild-type enzyme. The M151A/C295A/I86A mutant reacts optimally at 70 ºC with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3’- methylacetophenone by V115A/C295A/I86A, and (R)-1-phenylethanol from acetophenone by M151A/C295A/I86A mutant, in large-scale bioreduction processes.
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KEYWORD
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Computer-aided modeling, alcohol dehydrogenase, substrate specificity, asymmetric reduction, thermostability
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